Much of the progress in genetics and biotechnology has depended on the availability of techniques for amplifying nucleic acids to produce amounts amenable for analysis, for example, by sequencing, hybridization, fluorescent labeling, or the like. A wide variety of amplification techniques have been developed for this purpose, including polymerase chain reaction (PCR), ligase chain reaction (LCR), nucleic acid sequence-based amplification (NASBA), rolling circle, and the like.
PCR has been the most widely used method to amplify DNA from small quantities of DNA molecules. Although it is efficient and generates large quantities of DNA from small aliquots of DNA template, PCR always introduces biases when it is applied to amplify complex DNA libraries. A host of factors affect the relative amplification rates of individual sequences with a library, including the preference of primer annealing sites, the length and GC content differences of the template DNAs, non-specific priming at interior sites of sequences, temporal differences in extension times, and the like. Frequently, because of such factors, the polymerization, or primer extension, step is terminated prematurely by the denaturation step of the PCR; hence, smaller fragments and low GC content fragments are preferentially amplified.
It would be very useful for many applications where nucleic amplification plays a key role, if an amplification technique were available that minimized or eliminated the above short-comings of PCR.